The human distal gut microbiome is an extraordinary example of a mutualistic relationship wherein trillions of microbes ferment dietary and host-derived carbohydrates and the products of fermentation contribute to nutrient acquisition, gut epithelial health, and immune system development. The NIH-funded human microbiome project will generate neariy 1000 reference genomes from cultured and non-cultured microbes, and will supplement this data with DNA sequencing of microbial samples taken from human subjects. Glycoside hydrolases (GHs) comprise a significant proportion of the genes encoded by microbial genomes within the human distal gut microbiome and contribute to the depolymerization of recalcitrant dietary polysaccharides. A recent metagenomic analysis of the human distal gut microbiome revealed that of the 81 GH families present in the distal gut microbiome, GH family 3 was the most highly represented, which indicates that this gene family is important for carbohydrate utilization by the gut microflora. A number of different functional activities have been described for GH family 3 enzymes although the molecular determinants that define substrate specificity for these enzymes have not been elucidated. The long-term goal of the proposed research is to provide insight into the role of the GH family 3 genes in the metabolic repertoire of human gut microorganisms. In aim 1 we will characterize the substrate specificities of four GH farnily 3 enzymes from the bacterium Prevotella bryantii Bi4 using a library of natural plant cell wall derived oligosaccharides. In aim 2 we will employ a directed evolution approach for identifying amino acid residues that contribute to substrate specificity for one of these GH family 3 enzymes from P. bryantii Bi4. Results from the proposed studies will provide insight into the molecular determinants of substrate specificity for GH family 3 genes and will allow us to place this important gene family in the context of the metabolic repertoire of the gut-associated microflora. Public Health Relevance: The composition of microbial communities within the human colon is a critical factor that influences the development of numerous disease states including Crohn's disease and ulcerative colitis. This study will provide direct insight into the metabolic properties of the human colonic microflora. Understanding the metabolic characteristics of the gut microflora is essential for effective treatment of a wide variety of gastrointestinal disorders.